In vitro stimulation of reconstituted spleen B cells from GFP-SPL.

After 14 days of reconstitution, spleen B cells were enriched, as described in “Materials and methods.” Purified B cells were cultured in RPMI-1640 complete medium with various combinations of anti-μ Ab (10 μg/mL), LPS (20 μg/mL), rat antimouse CD40 mAb (1 μg/mL), mouse IL-4 (500 U/mL), or mouse IL-7 (100 U/mL). Cell proliferation by the same stimulatory reagents was examined by the incorporation of [³H]-TdR, as described in “Materials and methods.” As a control for GFP signal in B-lineage cells, we used the BM-derived pre-B cell lines from RAG1/GFP mice cultured in vitro using the method described by Whitelock and Witte.35Endogenous rag1 transcripts after stimulation of the spleen cells in vitro. Spleen cells from the GFP-SPL mice were stimulated in vitro as above, and the rag1 transcripts were monitored by RT-PCR with primers, as described in “Materials and methods.”